Use of photosensitisation

ABSTRACT

A composition comprising a conjugate of a photosensitiser and a bacteriophage is provided. The conjugate may be used to kill bacteria, particularly MRSA, EMRSA, VRSA, hetero-VRSA or CA-MRSA in a targeted method of photodynamic therapy.

The present invention relates to a composition comprising a conjugate ofa photosensitiser and a bacteriophage, particularly a staphylococcalbacteriophage, known as a staphylophage. The invention also relates tothe use of the conjugate in a method of photodynamic therapy forinfectious diseases.

BACKGROUND

The use of antimicrobial agents to counter bacterial infections isbecoming increasingly ineffective, due to the rapid emergence ofantibiotic resistance amongst many species of pathogenic bacteria. Onesuch pathogen is Staphylococcus aureus (S. aureus), whichcharacteristically causes skin infections such as boils, carbuncles andimpetigo, as well as infecting acne, burns and wounds. If the infectingorganism is a toxic strain, such infections, or colonised tampons, maygive rise to a life-threatening toxaemia known as toxic shock syndrome.The organism may also gain access to the bloodstream from theseinfections, or from foreign bodies such as intravenous catheters, and socause infections at other sites, such as endocarditis, osteomyelitis,meningitis and pneumonia

A number of bacteria are responsible for infection of skin and wounds,for example, coagulase-negative staphylococci, Staphylococcus aureus,streptococci, Corynebacterium spp., E. coli, Klebsiella aerogenes,Klebsiella pneumoniae, Enterobacter aerogenes, Propionibacterium acnes,Bacteroides spp., Pseudomonas aeruginosa and Peptostreptococcus spp.Increasingly, these bacteria are showing resistance to antibiotictreatment.

In particular, resistant strains of S. aureus have emerged.Methicillin-resistant S. aureus (MRSA) was first reported in 1961(Jevons, M. (1961) British Medical Journal, 1, 124-5), and these strainsare now a major cause of hospital-acquired infection throughout theworld, as well as being prevalent in many nursing and residential homes.This poses an alarming challenge to healthcare, causing significantinfection and morbidity of hundreds of patients in the UK each year(Ayliffe et al, J Hosp Infect (1988), 39, 253-90).

Since the first report of MRSA, these organisms have demonstratedresistance to a wide variety of antimicrobials including erythromycin,aminoglycosides, tetracyclines, trimethoprim, sulphonamides andchloramphenicol. MRSA strains have developed that are only susceptibleto a single class of clinically-available antibiotics: the glycopeptidessuch as vancomycin and teicoplanin. However, resistance is developingeven to these, as strains tolerant to vancomycin have now been reported(Hiramatsu, K. (1998) American Journal of Medicine, 104, 7S-10S). Thesestrains are variously known as VRSA (Vancomycin resistant Staphylococcusaureus) and hetero-VRSA (resistant strains arising from exposure to highlevels of vancomycin). At present, the management of patients with MRSAinfections usually involves the administration of antimicrobial agentsand again, there is evidence of the development of resistance to many ofthe agents used.

Due to the emergence of strains which are resistant to virtually allcurrently-available antimicrobials, MRSA is now a serious threat tohealth. The term MRSA itself now more accurately applies to methicillinand multiple antimicrobial-resistant S. aureus.

Certain strains of MRSA have been found to spread rapidly not onlywithin hospitals, but also between them. These strains have been termedepidemic MRSA (EMRSA). Since the first EMRSA strain (EMRSA-1) wasreported in 1981, 17 distinct EMRSA strains have been identified, all ofwhich are resistant to a number of antimicrobials. Recently, the twomost prevalent strains have been EMRSA-15 and -16, which account for60-70% of the 30000 MRSA isolates reported (Livermore, D (2000) Int. J.Antimicrobial Agents, 16, S3-S10). Importantly, strains of MRSA, (knownas community-acquired MRSA (CA-MRSA)) have also started to spread in thecommunity, ie. amongst non-hospitalised individuals.

It is clear from the above that alternative methods of counteringbacterial infection, particularly infection with MRSA, are urgentlyrequired.

One approach has been to employ a light-activated agent to achievelethal photosensitization of the organism. This involves treating theorganism with a light-activatable chemical (photosensitiser) which, uponirradiation with light of a suitable wavelength, generates cytotoxicspecies, resulting in bacteriolysis. This technique has been used toachieve killing of a wide range of bacteria, including S. aureus andMRSA strains, in vitro using toluidine blue O (TBO) and aluminiumdisulphonated phthalocyanine (AlPcS₂) as photosensitisers. Neitherphotosensitiser nor laser light alone exerted a bacteriocidal effect(Wilson et al, (1994) J Antimicrob Chemother 33, 619-24). In asubsequent study, 16 strains of EMRSA were found to be susceptible tokilling by low doses of red light (674 mm) in the presence of AlPcS₂(Griffiths et al, (1997) J Antirnicrob Chemother, 40, 873-6). At higherlight doses, 100% killing was achieved.

Photodynamic therapy (PDT) is the application of such an approach to thetreatment of disease. It is an established procedure in the treatment ofcarcinoma and forms the basis of a means of sterilising blood products.It has only been more recently that the application of PDT to thetreatment of infectious diseases has been evaluated. For example,haematoporphyrins in conjunction with an argon laser have been used totreat post-neurosurgical infections and brain abscesses (Lombard et al,(1985), Photodynamic Therapy of Tumours and other Diseases, Ed. Jori &Perria).

One potential problem associated with PDT of infectious diseases is itslack of specificity. Hence, if the photosensitiser binds to, or is takenup by, a host cell, as well as the target organism, then subsequentirradiation may also lead to the death of the host cell. A way toovercome this is by the use of targeting compounds: that is, anycompound that is capable of specifically binding to the surface of thepathogen.

Several targeting compounds have previously been shown to be successfulin eliminating specific strains of bacteria when they were conjugated toa photosensitiser. For example, immunoglobulin G (IgG) has been used totarget S. aureus Protein A (Gross et al (1997), Photochemistry andPhotobiology, 66, 872-8), monoclonal antibody against Porphyromonasgingivalis lipopolysaccharide (Bhatti et al (2000), Antimicrobial Agentsand Chemotherapy, 44, 2615-8) and poly-L-lysine peptides against P.gingivalis and Actinomyces viscosus (Soukos et al (1998), AntimicrobialAgents and Chemotherapy, 42, 2595-2601). A monoclonal antibodyconjugated via dextran chains to the photosensitiser tin (IV) chlorin e6(SnCe6) was selective for killing P. aeruginosa when exposed to light at630 nm, leaving S. aureus unaffected (Friedberg et al (1991), Ann N YAcad Sci, 618, 383-393).

The present inventors have used IgG conjugated to SnCe6 to target EMRSAstrains 1, 3, 15 and 16 (Embleton et al (2002), J Antimicrob Chemother,50, 857-864), achieving higher levels of killing than thephotosensitiser alone, and selectively killing the EMRSA strains in amixture with Streptococcus sanguis. However, a limitation of IgG is thatonly strains of S. aureus expressing Protein A can be targeted. Hencealternative targeting agents that can target any S. aureus strain aredesirable.

Bacteriophage are viruses that infect certain bacteria, often causingthem to lyse and hence effecting cell death. They have been proposed asantibacterial agents in their own right. However, one of the problemswith using staphylococcal bacteriophage (termed staphylophage) in thetreatment of S. aureus disease is their restricted host range. Althoughthere are polyvalent staphylophage which can lyse many S. aureusstrains, other strains are resistant and hence bacteriophages alonecould not provide an effective method of killing all strains of S.aureus.

It is known that although some bacteriophage will only kill a limitedrange of bacteria, they will bind to a broader range of bacteria. Thepresent inventors have now found that some bacteriophage can serve as aneffective, targeted delivery system for photosensitisers.

The present inventors have found that when a bacteriophage is linked toa photosensitiser, the photosensitiser-bacteriophage conjugate formed ishighly effective in killing bacteria when irradiated with light of asuitable wavelength.

Bacteriophage-photosensitiser conjugates could be used to treat orprevent a broad range of bacterial skin and wound infections. The mostfrequently isolated organisms from skin and wound infections are:coagulase-negative staphylococci, S. aureus, streptococci, e.g.Streptoccocus pyogenes, Corynebacterium spp., E coli, Klebsiellaaerogenes, Klebsiella pneumoniae, Enterobacter aerogenes,Propionibacterium acnes, Bacteroides spp., Pseudomonas aeruginosa andPeptostreptococcus spp.

In particular, conjugates of photosensitiser and staphylophage can beused in a method of photodynamic therapy against strains ofstaphylococci spp, particularly against MRSA, EMRSA, VRSA, hetero-VRSAand CA-MRSA.

The invention provides a composition comprising a photosensitizingcompound (photosensitiser) linked to a bacteriophage to form aphotosensitiser-bacteriophage conjugate. The bacteriophage may be astaphylococcal phage, and is preferably a staphylophage that can bind toStaphylococcus aureus, particularly MRSA, EMRSA, VRSA, hetero-VRSA orCA-MRSA. The composition may be used in a method of photodynamictherapy.

The bacteriophage is preferably linked to the photosensitiser using acovalent linkage. The photosensitiser and/or the bacteriophage containor may be modified to contain groups which can be covalently crosslinkedusing chemical or photoreactive reagents, to produce crosslinked bonds,for example thiol-thiol crosslinking, amine-amine crosslinking,amine-thiol crosslinking, amine-carboxylic acid crosslinking,thiol-carboxylic acid crosslinking, hydroxyl-carboxylic acidcrosslinking, hydroxyl-thiol crosslinking and combinations thereof.

The photosensitiser is suitably chosen from porphyrins (e.g.haematoporphyrin derivatives, deuteroporphyrin), phthalocyanines (e.g.zinc, silicon and aluminium phthalocyanines), chlorins (e.g. tin chlorine6, poly-lysine derivatives of tin chlorin e6, m-tetrahydroxyphenylchlorin, benzoporphyrin derivatives, tin etiopurpurin),bacteriochlorins, phenothiaziniums (e.g. toluidine blue, methylene blue,dimethylmethylene blue), phenazines (e.g. neutral red), acridines (e.g.acriflavine, proflavin, acridine orange, aminacrine), texaphyrins,cyanines (e.g. merocyanine 540), anthracyclins (e.g. adriamycin andepirubicin), pheophorbides, sapphyrins, fullerene, halogenated xanthenes(e.g. rose bengal), perylenequinonoid pigments (e.g. hypericin,hypocrellin), gilvocarcins, terthiophenes, benzophenanthridines,psoralens and riboflavin.

The invention is directed to killing bacteria using the above-describedconjugates. The bacteriophage used in the conjugate may be selectedaccording to the particular organism to be killed, in order to arrive atthe conjugate most effective against the particular infecting bacteria.In a preferred embodiment, the infecting bacterium is MRSA, EMRSA, VRSA,hetero-VRSA or CA-MRSA and the conjugate includes the staphylococcalphage 75 or phage φ11.

Table 1 below shows some examples of bacteria-bacteriophage pairs,although many more examples exist. Further novel bacteriophages can beisolated and/or adapted to the target bacteria. The specificity of thetreatment can be modified as required by using monovalentbacteriophages, polyvalent bacteriophages or combinations of monovalentbacteriophages or combinations of monovalent and polyvalentbacteriophages. TABLE 1 Bacterium Bacteriophage Staphylococcus aureus53, 75, 79, 80, 83, φ11, φ12, φ13, φ147, φMR11 Staphylococcusepidermidis 48, 71, numerous (182 different phage) Staphylococcus sppφ812, SK311, φ131, SB-I and U16 Streptococcus spp C₁, SF370.1, SP24,SFL, A1 (ATCC 12202-B1) various Corynebacterium spp φ304L φ304S, φ15,φ16, 782 Klebsiella aerogenes and P1clr100KM Klebsiella pneumoniae Ecoli P1, T1, T3, T4, T7 MS2 Enterobacter aerogenes Various, P1, M13Pseudomonas aeruginosa UNL-1, ACQ, UT1, tba1D3, E79, F8 & pf20 B3, F116,G101, B86; T7M, ACq, UT1, BLB, PP7 Propionibacterium acnes Various,including ATCC 29399-B1 Bacteroides spp B40-8 Numerous Gram-negativebacteria P1 Various

The composition of the invention suitably comprises at least 0.01 μg/ml,of the photosensitiser, preferably at least 0.02 μg/ml, more preferablyat least 0.05 μg/ml up to 200 μg/ml, preferably up to 100 μg/ml, morepreferably up to 50 μg/ml. The amount of the bacteriophage in thecomposition is suitably from 1×10⁵ to 1×10¹⁰ pfu, preferably from 1×10⁶to 1×10⁹ pfu, more preferably from 1×10⁶ to 1×10 ⁸ pfu.

The composition of the invention may further comprise a source ofdivalent ions, e.g. Ca²⁺ or Mg²⁺, preferably Ca²⁺. Examples includecalcium chloride, calcium carbonate and magnesium chloride. The ions aresuitably present in an amount of from 5 to 200 mM, preferably from 5 to15 mM, more preferably about 10 mM.

The composition may further comprise one or more ingredients chosen frombuffers, salts for adjusting the tonicity, antioxidants, preservatives,gelling agents and remineralisation agents.

The invention further provides a method of killing bacteria, comprising

(a) contacting an area to be treated with the composition of theinvention such that any bacteria in the area bind to thephotosensitiser-bacteriophage conjugate; and

(b) irradiating the area with light at a wavelength absorbed by thephotosensitiser.

Suitably the bacteria are as set out above in Table 1, preferablyStaphylococcus aureus, more preferably MRSA, EMRSA, VRSA, hetero-VRSA orCA-MRSA.

In the method of the invention, any light source that emits light of anappropriate wavelength may be used. The wavelength of the light isselected to correspond to the absorption maximum of the photosensitiserand to have sufficient energy to activate the photosensitiser. Thesource of light may be any device or biological system able to generatemonochromatic or polychromatic light. Examples include laser, lightemitting diode, arc lamp, halogen lamp, incandescent lamp or an emitterof bioluminescence or chemiluminescence. In certain circumstances,sunlight may be suitable. Preferably, the wavelength of the lightemitted by the light source may be from 200 to 1060 nm, preferably from400 to 750 nm. A suitable laser may have a power of from 1 to 100 mW anda beam diameter of from 1 to 10 mm. The light dose for laser irradiationis suitably from 5 to 333 J cm⁻², preferably from 5 to 30 J cm⁻² forlaser light. For white light irradiation, a suitable dose is from 0.01to 100 kJ/cm², preferably from 0.1 to 20 kJc/m², more preferably from 3to 10 kJ/Cm². The duration of irradiation is suitably from one second to15 minutes, preferably from 1 to 5 minutes.

The following light sources may be suitable for use in the presentinvention:

-   -   Helium neon (HeNe) gas laser (633 nm)    -   Argon-pumped dye laser (500-700 nm, 5W output)    -   Copper vapour-pumped dye laser (600-800 nm)    -   Excimer-pumped dye laser (400-700 nm)    -   Gold vapour laser (628 nm, 10 W output)    -   Tunable solid state laser (532-1060 nm), including Sd:YAG    -   Light emitting diode (LED) (400-800 nm)    -   Diode laser (630-850 nm, 25 W output), eg. gallium selenium        arsenide    -   Tungsten filament lamp    -   Halogen cold light source    -   Fluorescent lamp.

In the method of the invention, the composition is suitably in the formof a solution or a suspension in a pharmaceutically acceptable aqueouscarrier, but may be in the form of a solid such as a powder or a gel, anointment or a cream. The composition may be applied to the infected areaby painting, spreading, spraying or any other conventional technique.

The invention further provides the use of the composition for treatmentof the human or animal body. Suitably, the composition is provided foruse in the treatment of conditions resulting from bacterial infection,particularly by staphylococci, more particularly by MRSA, EMRSA, VRSA,hetero-VRSA or CA-MRSA.

The invention may be used to treat bacterial infection, particularly bystaphylococcal bacteria, more particularly by MRSA, EMRSA, VRSA,hetero-VRSA or CA-MRSA to treat or prevent skin infections such asboils, carbuncles, mastitis and impetigo, to treat or prevent infectionsof acne, burns or wounds, or to treat or prevent endocarditis,osteomyelitis, meningitis and pneumonia, arising as a result ofbacterial infection, to treat or prevent infections arising from the useof catheters, implants or other medical devices, or to prevent infectionfollowing an operation, such as a Caesarean section.

The invention may also be used in the prevention of carriage of thebacteria by carriers who themselves show few, if any, symptoms.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of a phage 75-SnCe6 conjugate on different EMRSAstrains.

FIG. 2 shows the effects of conjugate, no conjugate, photosensitiseronly or phage only and presence or absence of irradiation on EMRSA-16and S. epidermidis.

FIGS. 3 to 5 show the effect of the invention on EMRSA-16 and S. aureus8325-4, varying the light dose.

FIG. 6 shows the effect of light dose using a fixed concentration ofΦ11-SnCe6 conjugate on EMRSA-16.

FIG. 7 shows the effect of the invention on strains of VRSA (Mu3),hetero-VRSA (Mu50) and CA-MRSA (MW2).

FIG. 8 shows the effect of the invention on Streptococcus pyogenes.

FIG. 9 shows the effect of the invention on Propionibacterium acnes.

EXAMPLES

Materials and Methods

The following media were prepared:

Nutrient Broth 2 (NB2) Medium

One litre of medium was made by adding 25 g of Nutrient Broth 2 (Oxoid)(10.0 g/l Lab-Lemco powder, 10.0 g/l peptone, 5.0 g/l NaCl) to 1 litreof deionised, distilled water. After mixing, the medium was autoclavedat 121° C. for 15 min.

Tryptone Soya Yeast Broth (TSY)

One litre of medium was made by adding 39 g of Tryptone Soya Broth(Oxoid) (17.0 g/l pancreatic digest of casein, 3.0 g/l papaic digest ofsoybean meal, 2.5 g/l glucose, 2.5 g/l di-basic potassium phosphate, 5.0g/l NaCl) and 0.5% of yeast extract (9.8 g/l total nitrogen, 5.1 g/lamino nitrogen, 0.3 g/l NaCl) to 1 litre of deionised, distilled water.After mixing, the medium was autoclaved at 121° C. for 15 min.

Nutrient Broth 2 Top Agar

0.35% (w/v) of Agar Bacteriological (Agar No. 1, Oxoid) was added to NB2medium. After mixing, the medium was autoclaved at 121° C. for 15 min.

Nutrient Broth 2 Bottom Agar

0.7% (w/v) of Agar Bacteriological was added to NB2 medium. Afterautoclaving, 10 mM of CaCl₂ was added (10 ml 1M CaCl₂ in 1 litre ofNB2).

Columbia Blood Agar (CBA)

37.1 g of Columbia Agar Base (Oxoid) (23.0 g/l special peptone, 1.0 g/lstarch, 5.0 g/l NaCl, 10.0 g/l agar) was added to 1 litre of deionised,distilled water. After autoclaving, the liquid agar was allowed to coolat room temperature until cool enough to handle. 5% (v/v) defibrinatedhorse blood (E & O Laboratories, Scotland) was then added.

Mannitol Salt Agar (MSA)

111 g of Mannitol Salt Agar (Oxoid) (75.0 g/l NaCl, 10.0 g/l mannitol,1.0 g/l Lab-lemco powder, 10.0 g/l peptone, 0.025 g/l phenol red, 15.0g/l agar) was added to 1 litre of deionised, distilled water.

All mixtures were autoclaved at 121° C. for 15 min. The liquid agar wasthen poured into plates, covered and allowed to cool overnight.

Target Organisms

The organisms used in the examples were as follows, given as names andNCTC (National Collection of Type Cultures, UK) or ATCC (American TypeCulture Collection, USA) numbers:

-   Epidemic methicillin-resistant S. aureus (EMRSA)-1 (NCTC 11939)-   EMRSA-3 (NCTC 13130)-   EMRSA-15 (NCTC 13142)-   EMRSA-16 (NCTC 13143)-   Mu3 (ATCC 700698), is a methicillin-resistant Staphylococcus aureus    (MRSA) strain with heterogeneous resistance to vancomycin,    designated heterogeneously vancomycin-resistant Staphylococcus    aureus (hetero-VRSA) (Hanaki et al (1998). J. Anitimicrob.    Chemother. 42:199-209)-   Mu50 is the archetypal VRSA strain (Hiramatsu et al (1997). J.    Antimicrob. Chemother. 40:135-136)-   MW2 is a Community-acquired MRSA strain. Community acquired MRSA    strains (CA-MRSA) share the presence of staphylococcal cassette    chromosome mec (SCCmec) type IV in their genomes, are frequently    virulent, and predominantly cause skin and soft tissue infections.    The genome sequence of the prototypic CA-MRSA strain, MW2, has    revealed the presence of additional virulence factors not commonly    present in other S. aureus strains (Baba et al (2002), Lancet.    25;359(9320):1819-27).-   Staphylococcus epidermidis (NCTC 11047)-   Streptococcus pyogenes (ATCC 12202)-   Propionibacterium acnes (ATCC 29399)-   Staphyloccus aureus 8324-5 (Novick (1967) Virology 33; 156-166).

All were maintained by weekly subculture on CBA.

Bacteriophage

Phage 75 (Public Health Laboratory Service, UK) is a serogroup Fstaphylococcal phage, capable of infecting EMRSA-16, EMRSA-3 and weaklyinfecting EMRSA-15.

Bacteriophage φ11 (Iandolo et al, (2002), Gene 289 (1-2); 109-118) is atemperate bacteriophage of serological group B. φ11 is a transducingphage with a low lysogenisation frequency. It infects S.aureus lyticgroup III strains which include many human and animal pathogens.

Bacteriophage Propagation

Mid-exponential EMRSA-16 (300 μl) was added to 15 ml Falcon tubes.Approximately 10⁵ pfu of phage 75 were added to the tubes and allowed toincubate at room temperature for 30 min to allow the phage to infect thebacteria. 9 ml of cooled molten top NB2 agar (with 10 mM CaCl₂), wasadded to the tubes, and the mixture poured onto undried NB2 base agarplates. The plates were left to incubate at 37° C. overnight.

The next morning 1 ml of NB2 with 10 mM CaCl₂ was added to each plate,and the top agar with the liquid medium was scraped into a smallcentrifuge tube. The collected agar was then spun in a centrifuge at15000 rpm for 15 min at 4° C. The supernatant was collected and passedthrough a 0.45 μm (Nalgene) filter to remove any bacterial cells. Theresulting solution of phage 75 was stored at 4° C.

Bacteriophage Precipitation

Phage precipitation was carried out to purify the phage 75 from the NB2medium after propagation. To 5 ml of phage 75 in NB2, 1.3 ml of 5M NaCl(1M final concentration) and 0.2 ml 1× phosphate buffered saline (PBS)(8.0 g/l NaCl, 0.2 g/l KCl, 1.15 g/l Na₂HPO₄, 0.2 g/l KH₂PO₄) wereadded, and 20% PEG (polyethylene glycol 8000, Sigma) was added to thesolution and stirred slowly overnight until completely dissolved. Thesolution was then placed on ice overnight and the next morning thesolution was centrifuged at 8000 rpm for 20 min at 4° C. The supernatantwas removed and the remaining pellet was resuspended in 2.5 ml 1× PBS,and filtered through a 0.45 μm filter.

Photosensitiser

The photosensitiser used was tin (IV) chlorin e6 (SnCe6) (FrontierScientific, Lancashire, UK), which is photoactivatable at 633 nm.

Preparation of Conjugate

2 mg of SnCe6 was dissolved with stirring in 800 μl of activation buffer(0.1 M MES (2-(N-morpholino(ethanesulphonic acid) (Sigma)), 0.5 M NaCl,pH 5.5). An EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride) (Sigma) solution (4 mg in 1 ml activation buffer) and aS—NHS (N-hydroxysulphosuccinimide) (Fluka) solution (2.7 mg in 250 μlactivation buffer) were made.

To the dissolved SnCe6, 200 μl of dissolved EDC and S—NHS were added,and the mixture was left for 1 to 4 hours at room temperature withstirring to provide a stable amine-reactive intermediate. The mixturewas covered in aluminium foil as SnCe6 is a light sensitive reagent. Thereaction was quenched by adding 1.4 μl β-mercaptoethanol (Sigma).

Experiments were carried out using the reagents at a molar ratio ofSnCe6:EDC:S-NHS of 1:1:2.5.

The pH of the reactive SnCe6 mixture was neutralised to 7.0 by adding0.7 ml 1 M NaOH. 1.5 ml of phage 75 was then added to the amine-reactivesolution to allow the amino groups on the phage to react with thecarboxyl groups of the SnCe6, and then mixed for 4 to 16 hours. Thereaction was quenched with 2.5 μl ethanolamine (Sigma).

The photosensitiser-phage conjugate (PS-phage) was separated from freePS after conjugation by precipitating the PS-phage twice, as describedabove in Bacteriophage Precipitation. The PS-phage was then dialysedagainst PBS.

In the examples below, the concentration of phage 75 is 7.3×10⁶ pfu/mland the concentration of SnCe6/bacteriophage-SnCe6 is 1.5 μg/ml.

Laser

The laser used was a Model 127 Stabilite helium-neon (He/Ne) laser(Spectra Physics, USA) with a power output of 35 mW. The laser emittedradiation in a collimated beam, diameter 1.25 mm, with a wavelength of633 nm.

Example 1

A culture of EMRSA-16 in the mid-exponential growth phase was diluted to1×10⁷ cfu/ml. 20 μl samples of the diluted bacteria were then placedinto wells of a 96-well plate (Nunc), together with a magnetic stirrerbar.

100 μl of the phage 75-SnCe6 conjugate prepared above and calciumchloride (CaCl₂) to a final concentration of 10 mM was added to thebacteria. The contents of the wells were left to incubate at roomtemperature for 5 min, with stirring. Controls were performed with 100μl 1×PBS added to the bacteria and used as a reference for experimentalsamples. The experiment was carried out in duplicate.

After incubation, the contents of the well were directly exposed to thelaser light for 5 min, with stirring, corresponding to an energy densityof 21 J/cm². Aluminium foil was placed in the surrounding wells to allowany escaping laser light to be reflected back into the target well.Controls were performed with no laser irradiation.

After exposure to the laser, 100 μl samples were immediately taken fromeach well and serially diluted, from 10⁻¹ to 10⁻⁴, in 1 ml TSY in 1.5 mlEppendorf tubes. Aliquots of 50 μl of each dilution were then placed andspread out on half a CBA plate. The plates were placed in a 37° C.incubator overnight. The following morning the number of survivors wascounted, the average between the four sets was taken and multiplied bythe appropriate dilution factor, and graphically analysed.

Phage at 7.3×10⁶ pfu/ml

SnCe6/phage at 1.5 μg/ml

It was found that over 99.9% of the EMRSA-16 were killed.

Example 2

Example 1 was repeated, using EMRSA-1 in place of EMRSA-16. It was foundthat 99.98% of the bacteria were killed.

Example 3

Example 1 was repeated, using EMRSA-3 in place of EMRSA-16. It was foundthat over 99.99% of the bacteria were killed.

Example 4

Example 1 was repeated, using EMRSA-15 in place of EMRSA-16. It wasfound that over 99.99% of the bacteria were killed.

Example 5

Example 1 was repeated, using S. epidermidis in place of EMRSA-16. Itwas found that over 99.99% of the bacteria were killed.

Result for Examples 1 to 5 are presented in FIG. 1.

Example 6

Example 1 was repeated, using 10 μl each EMRSA-16 and S. epidermidis inplace of the 20 μl samples of EMRSA-16. Samples were plated on MBAplates for enumeration.

Phage at 7.3×10⁶ pfu/ml

SnCe6/phage at 1.5 μg/ml

21 J/cm² laser light

It was found that over 99.99% of both bacterial strains were killed inthe mixed culture.

Comparative Example

Example 6 was repeated, firstly in the absence of conjugate, and withoutexposing to laser light, secondly with SnCe6 photosensitiser andexposure to laser light, and thirdly with phage 75 and without exposureto laser light.

The results for Example 6 and for the Comparative Example are presentedin FIG. 2.

The Examples show that the conjugate is highly effective at killing allof the EMRSA strains tested. Since phage 75 is only capable of infectingEMRSA-15 and EMRSA-16, this indicates that the phage is able tosuccessfully bind to strains it is incapable of infecting, thus actingas an effective targetting agent. The attached photosensitisers theneffected the killing upon laser irradiation.

Significant kills were also obtained with S. epidermidis, both alone andin a mixture with MRSA, indicating that the phage also bound tonon-related staphylococcal strains. The phage 75-SnCe6 conjugate isuseful for a variety of staphylococcal infections.

Example 7

Targeted Photodynamic Therapy Using Φ11-SnCe6 Conjugates AgainstStaphylococcus aureus and a Laser Light Source

Bacteriophage Φ11 was propagated and precipitated as described above forphage 75, except that S aureus strain 8325-4 was used as the propagatingstrain. Tin chlorin e6 (SnCe6) was conjugated onto Staphylococcus phageΦ11 using the method described above, achieving bound concentrations of2.3 and 3.5 μg ml⁻¹ SnCe6 with the phage Φ11 at 4.7×10⁷ pfu.ml⁻¹. TheseΦ11-SnCe6 conjugates were then incubated with various strains ofStaphylococcus aureus and exposed to laser light at 633 nm from a 35 mWHeNe laser (21 J/cm²) for 5 minutes. The final concentration ofconjugated SnCe6 was 1.15 μg ml⁻¹.

The results show that Φ11-SnCe6 conjugates achieved a 92.33% kill of S.aureus 8325-4 (compared to control counts in phosphate buffered saline)after 5 minutes exposure, whilst SnCe6 at a corresponding concentration(1.15 μg ml⁻¹) did not achieve any kill. The results are presented inFIG. 3.

We have also shown that this Φ11-SnCe6 conjugate is effective against amethicillin-resistant strain of the organism (EMRSA-16), achieving88.11% kill, even though Φ11 only infects this strain under stringentoptimal conditions. A range of control experiments such as; lightwithout photosensitiser (L+S−), photosensitiser without light (L−S+),and unconjugated phage at 1×10⁷ pfu ml⁻¹ (L−S−); did not result insignificant kills. The results are presented in FIG. 4.

By increasing the light dose to 10 minutes in the presence of calcium(10 mM) we are now achieving 99.88% kills against S. aureus 8325-4 usingΦ11-SnCe6 conjugates (1.75 μg ml⁻¹). The results are presented in FIG.5.

For FIGS. 3 to 5 the photosensitiser (either SnCe6 or Φ11-SnCe6) wasadded to give a final concentration of 1.15 μg ml⁻¹ (with respect toSnCe6). The light source was a 35 mW Helium/Neon laser and irradiation(when used) was for 5 minutes in the case of FIGS. 3 and 4, and for 10minutes in the case of FIG. 5.

The effect of varying the light dose on the kills obtained with theSnCe6-phage Φ11 conjugate was investigated. The experiments were carriedout as described above except that the bacterial suspensions wereexposed to light from the Helium/Neon laser for different periods oftime—these were 1, 5, 10, 20 and 30 minutes. In each case, theconcentration of the Φ11-SnCe6 conjugate (final concentration equivalentto 3.5 μg ml⁻¹ of SnCe6) was the same.

Incubation of the organism with the Φ11-SnCe6 conjugate for up to 60minutes in the dark had no significant effect on the viable count.However, significant reductions in the viable count were obtained whenthe suspensions were exposed to laser light in the presence of theΦ11-SnCe6 conjugate—greater kills were obtained with the longer exposuretimes. Using an exposure time of 30 minutes, a reduction in the viablecount of approximately 99.9999% was obtained. Φ11-SnCe6 was used to givea final concentration of 3.5 μg ml⁻¹ (with respect to SnCe6). The lightsource was a 35 mW Helium/Neon laser and irradiation (when used) was for1, 5, 10, 20 or 30 minutes. The results are presented in FIG. 6.

In FIGS. 3 to 6

-   SnCe6=tin chlorin e6-   Φ11-SnCe6=tin chlorin e6 conjugated to bacteriophage Φ11-   PBS=Phosphate buffered saline-   L+S+=bacteria irradiated in the presence of conjugate-   L+S−=bacteria irradiated in the absence of conjugate-   L−S+=bacteria exposed to conjugate in the absence of light-   L−S−=bacteria exposed neither to light nor conjugate

Example 8

Lethal Photosensitisation of Staphylcoccus aureus Using a phage 75-Tin(IV) Chlorin e6 Conjugate and a White Light Source

Bacterial strains: S. aureus 8325-4

-   -   EMRSA-16

Light source: KL200 (Schott). This is a 20-watt halogen cold lightsource. The light guide attached to it is a flexible optic fibre bundlewhich is directed onto a 96 well plate at a distance of 5 cm. A squareof 4-wells is placed at the centre of the light source.

Approx light intensity=44,000 lux or 470 μW/nm

Phage 75 was conjugated to SnCe6 as described above. Phages were used ata concentration of 1×10⁷ pfu/ml.

Overnight cultures of S. aureus grown in nutrient broth werecentrifuged, resuspended in PBS and adjusted to an OD of 0.05 at 600 nm(approximately 4×10⁷ cfu/ml)

50 μl of bacterial culture was aliquoted into a 96-well plate and 50 μlof the one of the following solutions added to the wells:

-   1) 3.5 μg/ml SnCe6-phage 75 (final concentration 1.75 μg/ml, 1×10⁶    pfu/well) in PBS-   2) 1.75 μg/ml SnCe6-phage 75 (final concentration 0.875 μg/ml, 5×10⁵    pfu/well) in PBS-   3) 3.5 μg/ml SnCe6 in PBS (final concentration 1.75 μg/ml)-   4) 1.75 μg/ml SnCe6 in PBS (final concentration 0.875 μg/ml)-   5) PBS-   6) Phage 75 at a concentration of 5×10⁵ or 1×10⁶ pfu/well in PBS

Wells were either exposed to white light (4 wells at a time) or wrappedin tin foil and stored in the dark.

After various exposure times an aliquot was taken from each well,serially diluted and spread onto Columbia blood agar. Agar plates wereincubated overnight at 37° C. and counted the next day.

Results TABLE 2 Final concentration of Exposure L+ S+ SnCe6 L+S+ phage75-SnCe6 photosensitiser time % kill % kill S. aureus 8325-4  1.75 μg/ml10 min  97.8%  99.96% 0.875 μg/ml 10 min  45.3%  98.98%  1.75 μg/ml 20min  97.9% 99.998% EMRSA-16  1.75 μg/ml 10 min    0%  99.75% 0.875 μg/ml10 min    0%  99.69%  1.75 μg/ml 20 min 99.78% 99.997%% kill - this is calculated compared to bacteria incubated with PBS andkept in the dark All results are the average of replicate experiments.Controls included bacteria incubated with SnCe6, phage 75-SnCe6 andphage 75 without exposure to white light. Phage 75 was also exposed towhite light. All controls had bacterial counts which were notsignificantly different to the control suspension which had nophotosensitiser added and was not irradiated.

Example 9

Further tests were carried out on S. aureus strains Mu3, Mu50 and MW2.To suspensions of vancomycin-resistant strains of Staphylococcus aureus(Mu3 and Mu50) or a community-acquired strain of MRSA (MW2), saline,phage 75, SnCe6 or phage 75-SnCe6 was added and samples exposed to lightfrom a 35 mW Helium/Neon laser.

The concentration of SnCe6 used was 1.5 μg/ml, the phage concentrationwas 5.1×10⁷ plaque-forming units/ml and the light energy dose was 21J/cm². The numbers above the bars represent the % kill of the organismrelative to the sample to which saline only was added. The results arepresented in FIG. 7.

Example 10

Lethal Photosensitization of Streptococcus pyogenes Using Tin chlorin e6(SnCe6).

streptococcus pyogenes ATCC 12202 was grown in Brain Heart Infusionbroth at 37° C. in an atmosphere consisting of 5% CO₂ in air. The cellswere harvested by centrifugation and re-suspended in phosphate bufferedsaline (PBS) and diluted to 1×10⁷ cfu/ml in PBS. 20 μl samples of thediluted bacterial suspension were then placed into wells of a 96-wellplate, together with a magnetic stirrer bar. 100 μl of differentconcentrations (1-50 μg/ml) of the SnCe6 in PBS was added to thebacterial suspensions. Controls were performed with 100 μl PBS added tothe bacteria and either irradiated (L+S−) or kept in the dark (L−S−).The experiment was carried out in duplicate.

After incubation, the contents of some of the wells were exposed tolight from the 35 mW Helium/Neon laser emitting light with a wavelengthof 633nm for 10 min, with stirring, corresponding to an energy densityof 42 J/cm². Aluminium foil was placed in the surrounding wells to allowany escaping laser light to be reflected back into the target well.Control wells were not irradiated with laser light.

After exposure to the laser light, 100 μl samples were immediately takenfrom each well and serially diluted, from 10⁻¹ to 10⁻⁵, in 1 ml TSY in1.5 ml Eppendorf tubes. Duplicate 50 μl aliquots of each dilution werethen spread out on half a CBA plate. The plates were placed in a 37° C.incubator for up to 48 h and the resulting colonies were counted todetermine the number of surviving organisms.

Incubation of the organism in the dark with increasing concentrations ofSnCe6 had no significant effect on the viable count. Neither didirradiation of the organism with laser light in the absence of thephotosensitiser. However, irradiation of the organism in the presence ofSnCe6 resulted in a concentration-dependent decrease in the viablecount. A 99.9997% kill of the organism was obtained using aphotosensitiser concentration of 50 μg/ml. The results are presented inFIG. 8. In FIG. 8

L+(open bars)=cultures irradiated with laser light in the absence ofSnCe6 as well as in the presence of various concentrations of thephotosensitiser,

L−(shaded bars)=cultures incubated in the dark in the absence of SnCe6as well as in the presence of various concentrations of thephotosensitiser.

Example 11

Lethal Photosensitization of Propionibacterium acnes Using Tin Chlorine6 (SnCe6).

Propionibacterium acnes ATCC 29399 was grown in pre-reduced Brain HeartInfusion broth at 37° C. in an anaerobic atmosphere. The cells wereharvested by centrifugation and re-suspended in phosphate bufferedsaline (PBS) and diluted to 1×10⁸ cfu/ml in PBS. 20 μl samples of thediluted bacterial suspension were then placed into wells of a 96-wellplate, together with a magnetic stirrer bar. 100 μl of differentconcentrations (1-50 μg/ml) of the SnCe6 in PBS was added to thebacterial suspensions. Controls were performed with 100 μl PBS added tothe bacteria and either irradiated (L+S−) or kept in the dark (L−S−).The experiment was carried out in duplicate.

After incubation, the contents of some of the wells were exposed tolight from the 35 mW Helium/Neon laser emitting light with a wavelengthof 633 nm for 10 min, with stirring, corresponding to an energy densityof 42 J/cm². Aluminium foil was placed in the surrounding wells to allowany escaping laser light to be reflected back into the target well.Control wells were not irradiated with laser light.

After exposure to the laser light, 100 μl samples were immediately takenfrom each well and serially diluted, from 10⁻¹ to 10⁻⁵, in 1 ml ofpre-reduced TSY in 1.5 ml Eppendorf tubes. Duplicate 50 μl aliquots ofeach dilution were then spread out on half a CBA plate. The plates wereincubated anaerobically at 37° C. and the resulting colonies werecounted to determine the number of surviving organisms.

Incubation of the organism in the dark with increasing concentrations ofSnCe6 had no significant effect on the viable count. Neither didirradiation of the organism with laser light in the absence of thephotosensitiser. However, irradiation of the organism in the presence ofSnCe6 resulted in a concentration-dependent decrease in the viablecount. A 100% kill of the organism was obtained using a photosensitiserconcentration of 50 μg/ml. The results are presented in FIG. 9. In FIG.9

L+(open bars)=cultures irradiated with laser light in the absence ofSnCe6 as well as in the presence of various concentrations of thephotosensitiser;

L−(shaded bars)=cultures incubated in the dark in the absence of SnCe6as well as in the presence of various concentrations of thephotosensitiser.

Example 12

Preparation of Conjugate of TBO and Bacteriophage

1 mg of toluidine blue O (TBO) was dissolved in 800 μl of activationbuffer (0.1M MES, 0.5M NaCl pH5.5) together with 0.4 mg EDC and 0.6 mgof S—NHS and 200 μl of phage (5×10⁷ pfu/ml). The reaction was allowed toproceed for 15 to 30 minutes with stirring after which time the EDC wasneutralised by adding 1.4 μl of 2-mercaptoethanol. The reaction wasallowed to proceed for a further 2 to 4 hours after which time thereaction was quenched by adding hydroxylamine to a final concentrationof 10 mM.

The TBO-phage conjugate was separated from free TBO by two rounds ofphage precipitation followed by dialysis against PBS.

1. A composition comprising a conjugate of a photosensitiser and abacteriophage.
 2. A composition according to claim 1, wherein thebacteriophage is a staphylococcal bacteriophage.
 3. A compositionaccording to claim 1, wherein the photosensitiser is covalently linkedto the bacteriophage.
 4. A composition according to claim 1, wherein thephotosensitiser is chosen from Porphyrins, phthalocyanines, chlorins,bacteriochlorins, phenothiaziniums, phenazines, acridines, texaphyrins,cyanines, anthracyclins, pheophorbides, sapphyrins, fullerene,halogenated xanthenes, perylenequinonoid pigments, gilvocarcins,terthiophenes, benzophenanthridines, psoralens and riboflavin.
 5. Acomposition according to claim 4, wherein the photosensitiser is tin(IV) chlorin e6 (SnCe6).
 6. A composition according to claim 1, whereinthe bacteriophage is chosen from phage 53, 75, 79, 80, 83, φ11, φ12,φ13, φ147, φMR11, 48, 71, φ812, SK311, φ131, SB-I, U16, C₁, SF370.1,SP24, SFL, A1, ATCC 12202-B1, f304L, φ304S, φ15, φ16, 782, P1c1r100KM,P1, T1, T3, T4, T7 MS2, P1, M13, UNL-1, ACQ, UT1, tba1D3, E79, F8, pf20B3, F116, G101, B86, T7M, ACq, UT1, BLB, PP7, ATCC 29399-B1 and B40-8.7. A composition according to claim 6, wherein the bacteriophage isphage 75 or phage Φ11.
 8. A composition according to claim 1, whereinthe concentration of the photosensitiser is from 0.01 to 200 μg/ml.
 9. Acomposition according to claim 1, wherein the concentration of thebacteriophage is from 1×10⁵ to 1×10¹⁰ pfu/ml.
 10. A compositionaccording to claim 1, which further comprises a source of Ca²+ ions,preferably calcium chloride.
 11. A composition according to claim 1, inthe form of a solution in a pharmaceutically acceptable carrier.
 12. Acomposition according to claim 1, wherein the composition furthercomprises one or more of a buffer, salt, antioxidant, preservative,gelling agent or remineralisation agent.
 13. A method of killingbacteria, comprising (a) contacting an area to be treated with acomposition according to claim 1, such that any bacteria present bind tothe photosensitiser-bacteriophage conjugate; and (b) irradiating thearea with light at a wavelength absorbed by the photosensitiser.
 14. Amethod according to claim 13, wherein the bacteria are staphylococcus,particularly MRSA, EMRSA VRSA, hetero-VRSA or CA-MRSA.
 15. A methodaccording to claim 13, wherein the light is laser light or white light.16. A method according to claim 15, wherein the laser light is from ahelium neon gas laser.
 17. A method according to claim 15, wherein thelaser light has a wavelength of from 200 to 1060 nm.
 18. A methodaccording to claim 15, wherein the laser has a power of from 1 to 100 mWand a beam diameter of from 1 to 10 mm.
 19. A method according to claim18, wherein the light dose of laser irradiation is from 5 to 333 Jcm⁻².20. A method according to claim 15, wherein the light dose of whitelight is from 0.01 to 100 J/cm².
 21. A method according to claim 15,wherein the duration of irradiation is form one second to 15 minutes.22. A method according to claim 13, wherein the composition is presentin or on the area to be treated at a concentration of from 0.00001 to 1%w/v.
 23. Use of a composition according to claim 1, for treatment of thehuman or animal body.
 24. Use of a composition according to claim 1, inthe manufacture of a medicament for treatment of bacterial infection.25. Use according to claim 24, wherein the bacterial infection is S.aureus, particularly MRSA, EMRSA, VRSA, hetero-VRSA or CAMRSA.
 26. Useof a bacteriophage as a targeting agent in photodynamic therapy (PDT).27. Use according to claim 26, wherein the bacteriophage is astaphylococcal phage.
 28. A composition according to claim 1,substantially as described in the Examples.
 29. A method according toclaim 13, substantially as describe in the Examples.
 30. A use accordingto claim 23, substantially as described in the Examples.